1. Field of the Invention
The present invention relates to an improved strain of Rhodotorula graminis, which is a yeast strain capable of producing phenylalanine ammonia lyase (PAL), useful for the manufacture of L- phenylalanine from t-cinnamate.
2. Brief Description of the Background Art
L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.), is an enzyme found in several plants and also in yeast. It catalyzes the deamination of L-phenylalanine to give trans-cinnamic acid. In 1981, Yamada et al in Applied and Environmental Biology, 42:773-778 (1981) described the use of
for the rapid conversion of trans-cinnamic acid to L-phenylalanine, with a conversion yield of above 70%. This publication opened the way to investigate and apply commercially, the use of PAL for the production of phenylalanine.
The enzyme has been isolated and purified from a variety of organisms, especially the yeast Rhodotorula glutinis (see for example Hodgkins, J. Biol. Chem. 246 2977-2985 (1971), or Fritz, R. R. et al, ibid: 251: 4646-4650 (1976)).
It has been proposed that the purified enzyme is suitable for treatment of mouse neoplastic tumors (Abell, C. W., et al Cancer Res., 33: 2529-2532 (1973)), the analyses of serum phenylalanine in monitoring patients with phenylketonuria (PKU) (Chen, R. et al, Science, 197: 665-667 (1977)), or the depletion of serum phenylalanine using immobilized PAL (Ambrus, C. M. et al., Science, 201: 837-839 (1978)). However, by far the greatest application of PAL to date is in the commercial production of phenylalanine from ammonia and t-cinnamate.
In 1981, Marusich, W. C. et al, J. Bacteriol., 146: 1013-1019 (1981) showed that the highest specific activity of PAL could be obtained during mid exponential growth of Rhodotorula glutinis in a defined medium containing L-phenylalanine as the sole source of carbon. Nakamichi, K. et al., Eur. J. Appl. Microbiol. Biotechnol. 18: 158-162 (1983) confirmed that PAL activity in Rhodotorula glutinis was induced by the addition of L-phenylalanine, and showed that the activity reached a maximum after about six hours of induction and then diminished gradually. The enzyme was also induced by D-phenylalanine, L-isoleucine, D-isoleucine, L-leucine, D-leucine, L-valine, L-methionine, L-tryptophan, and L-tyrosine. When 0.1% L-isoleucine was added, high PAL activity was substained for a relatively long time. This publication also demonstrated that cells induced with isoleucine in combination with D-leucine or L-valine increased PAL specific activity over isoleucine alone.
PAL, however is also rapidly degraded in the absence of the inducer during fermentation and has a half-life of approximately 2-5 hours during fermentations of most Rhodotorula rubra strains.
McGuire, U.S. Pat. No. 4,598,047 shows mutant strains of Rhodotorula rubra (GX 5902, GX 5903, GX 5904 specifically) useful for PAL production.
Rhodotorula graminis wild strain GX 5007 a soil isolate, (Durham, et al., J. Bact. 160:771-777 (1984); this publication refers to GX 5007 as "KGX 39") has PAL activity. GX 5007 has several advantages over other production strains of Rhodotorula rubra. It grows 15-20% faster and requires less yeast extract, has no L-methionine requirement during induction, and PAL half-life during fermentation is about 9 hours. R. graminis GX 5007, however, is undesirable as a production strain due to low PAL titers obtained during fermentation.
A need therefore continues to exist for commercially useful production strains of PAL, which can be utilized in the preparation of PAL. Also, a need for ever more efficient methods of induction of PAL activity in Rhodotorula strains is needed.